Stuart Ibsen

Microbubble-mediated ultrasound therapy: a review of its potential in cancer treatment

The inherently toxic nature of chemotherapy drugs is essential for them to kill cancer cells but is also the source of the detrimental side effects experienced by patients. One strategy to reduce these side effects is to limit the healthy tissue exposure by encapsulating the drugs in a vehicle that demonstrates a very low leak rate in circulation while simultaneously having the potential for rapid release once inside the tumor. Designing a vehicle with these two opposing properties is the major challenge in the field of drug delivery. A triggering event is required to change the vehicle from its stable circulating state to its unstable release state. A unique mechanical actuation type trigger is possible by harnessing the size changes that occur when microbubbles interact with ultrasound. These mechanical actuations can burst liposomes and cell membranes alike allowing for rapid drug release and facilitating delivery into nearby cells. The tight focusing ability of the ultrasound to just a few cubic millimeters allows for precise control over the tissue location where the microbubbles destabilize the vehicles. This allows the ultrasound to highlight the tumor tissue and cause rapid drug release from any carrier present. Different vehicle designs have been demonstrated from carrying drug on just the surface of the microbubble itself to encapsulating the microbubble along with the drug within a liposome. In the future, nanoparticles may extend the circulation half-life of these ultrasound triggerable drug-delivery vehicles by acting as nucleation sites of ultrasound-induced mechanical actuation. In addition to the drug delivery capability, the microbubble size changes can also be used to create imaging contrast agents that could allow the internal chemical environment of a tumor to be studied to help improve the diagnosis and detection of cancer. The ability to attain truly tumor-specific release from circulating drug-delivery vehicles is an exciting future prospect to reduce chemotherapy side effects while increasing drug effectiveness.

A novel nested liposome drug delivery vehicle capable of ultrasound triggered release of its payload.

The use of focused ultrasound can be an effective method to locally highlight tumor tissue and specifically trigger the activation of echogenic drug delivery vehicles in an effort to reduce systemic chemotherapy side effects. Here we demonstrate a unique ultrasound triggered vehicle design and fabrication method where the payload and a perfluorocarbon gas microbubble are both encapsulated within the internal aqueous space of a liposome. This nested lipid shell geometry both stabilized the microbubble and ensured it was spatially close enough to interact with the liposome membrane at all times. The internal microbubble was shown to fragment the outer liposome membrane upon exposure to ultrasound at intensities of 1-1.5MPa. The focused ultrasound allowed the release of the internal payload to localized regions within tissue phantoms. The vehicles showed high payload loading efficiency of 16%, stability in blood of several hours, and low level macrophage recognition in vitro. High speed fluorescent videos present the first optical images of such vehicles interacting with ultrasound. This ability to open the outer membrane in small regions of deep tissue could provide a second level of spatial and temporal control beyond biochemical targeting, making these particles promising for in vivo animal studies.

Changes in signal parameters over time for an echolocating Atlantic bottlenose dolphin performing the same target discrimination task.

This study documents the changes in peak frequency, source level, and spectrum shape of echolocation clicks made by the same dolphin performing the same discrimination task in 1998 and in 2003/2004 with spherical solid stainless steel and brass targets. The total average peak frequency used in 1998 was 138 kHz but in 2003/2004 it had shifted down nearly 3.5 octaves to 40 kHz. The total average source level also shifted down from 206 dB in 1998 to 187 kHz in 2003/2004. The standard deviation of these parameter values within time periods was small indicating a consistent difference between time periods. The average parameter values for clicks used when exposed to brass versus steel targets were very similar indicating that target type did not greatly influence the dolphins average echolocation behavior. The spectrum shapes of the average clicks used in 1998 and in 2003/2004 were nearly mirror images of each other with the peak energy in 2003/2004 being concentrated where the 1998 clicks had the lowest energy content and vice versa. Despite the dramatic differences in click frequency content the dolphin was able to perform the same discrimination task at nearly the same level of success.

Functional bandwidth of an echolocating Atlantic bottlenose dolphin (Tursiops truncatus)

The frequency band that an Atlantic bottlenose dolphin (Tursiops truncatus) used to perform an echolocation target discrimination task was determined using computer simulated phantom targets. The dolphin was trained to discriminate frequency filtered phantom targets from unfiltered ones in a go/no-go paradigm. The dolphins performance indicated perception of echo alteration only when applied filters interfered with the frequency band between 29 and 42 kHz. The dolphin did not behaviorally convey perception of applied filters that affected frequencies outside this functional bandwidth, such as a low pass 43 kHz or a high pass 28 kHz filter. The upper limit of the functional bandwidth at 42 kHz corresponded with the dolphins upper hearing limit of 45 kHz, as determined through auditory evoked potential measurements. The lower limit of the functional bandwidth corresponded to a drop in intensity below 30 kHz within the dolphins echolocation clicks. The randomized presentation of different filters showed that the dolphin paid attention to the entire 29-42 kHz band for each trial, not just subsets. The absence of temporal cues between some of the targets the dolphin could discriminate indicated that in these cases the target discrimination cues were based solely on the frequency content.

Fluorescent Microscope System to Monitor Real-Time Interactions between Focused Ultrasound, Echogenic Drug Delivery Vehicles, and Live Cell Membranes

Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5MPa the membranes were shown to completely fragment while at intensities below 1MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 μL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology

The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications.

Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX‐PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber‐optic LED system. Pharmacokinetic studies showed that DOX‐PCB had an α circulation half‐life of 10 min which was comparable to that of DOX at 20 min. DOX‐PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX‐PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.

Ultrasound Mediated Localized Drug Delivery

Chemotherapy is one of the frontline treatments for cancer patients, but the toxic side effects limit its effectiveness and potential. The goal of drug delivery is to reduce these side effects by encapsulating the drugs in a carrier which prevents release and can circulate throughout the body causing minimal damage to the healthy tissue. Slow release carriers have been developed which reduce the exposure to healthy tissue but this slow release also limits the maximum levels of drug in the tumor and nonspecific accumulation in healthy tissue remains a major hurdle. The next advance is to design these carriers to produce a rapid burst release of drug, but only in response to a localized trigger. The trigger of choice is low intensity focused ultrasound. A new particle is described here which incorporates an ultrasound sensitive microbubble of perfluorocarbon gas within a protective liposome carrier along with the payload. It is shown that this design can accomplish the desired burst release when exposed to ultrasound focused to small spatial locations within tissue phantoms. The ability to trigger release could provide a second level of spatial and temporal control beyond biochemical targeting or passive accumulation, making these promising particles for further development.

Extraction Protocol and Mass Spectrometry Method for Quantification of Doxorubicin Released Locally from Prodrugs in Tumor Tissue

The localized conversion of inactive doxorubicin prodrug chemotherapeutics to pharmacalogically active forms is difficult to quantify in mouse tumor models because it occurs only in small regions of tissue. The tumor tissue extraction protocol and LC-MS/MS analysis method described here were optimized to obtain a detection limit of 7.8 pg for the activated doxorubicin and 0.36 ng for the doxorubicin prodrug. This method can be useful for determining the biodistribution and activation efficiency for many different doxorubicin prodrugs. It can also be used for quantification of doxorubicin from tumor models that have poor vascularization resulting in low tissue accumulation.